Pumpkin line HWN 130-1039T

ABSTRACT

The invention provides seed and plants of the pumpkin line designated HWN 130-1039T. The invention thus relates to the plants, seeds and tissue cultures of pumpkin line HWN 130-1039T, and to methods for producing a pumpkin plant produced by crossing a plant of pumpkin line HWN 130-1039T with itself or with another pumpkin plant, such as a plant of another line. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of pumpkin line HWN 130-1039T, including the fruit and gametes of such plants.

This application claims the priority of U.S. Provisional Appl. Ser. No.60/968,871, filed Aug. 29, 2007, the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of plant breeding and, morespecifically, to the development of Cucurbita pepo Halloween typepumpkin line HWN 130-1039T.

2. Description of Related Art

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include greateryield, resistance to insects or pests, tolerance to heat and drought,better agronomic quality, higher nutritional value, growth rate andfruit properties.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different varieties produces auniform population of hybrid plants that are heterozygous for many geneloci. Conversely, a cross of two plants each heterozygous at a number ofloci produces a population of hybrid plants that differ genetically andare not uniform. The resulting non-uniformity makes performanceunpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines are developed byselfing and selection of desired phenotypes. The new lines are evaluatedto determine which of those have commercial potential.

One crop species which has been subject to such breeding programs and isof particular value is the pumpkin. Pumpkin is one common name formembers of the genus Cucurbita of the family Cucurbitaceae which areharvested as fully mature fruits. Cucurbita species include Cucurbitapepo, Cucurbita maxima, Cucurbita mixta, and Cucurbita moschata. In theUnited States, the thick-growing, small-fruited bush, or non-trailingvarieties of C. pepo grown for immature fruit harvest are called summersquash or zucchini, and the long-season, long-trailing, large-fruitedvarieties are called pumpkin or winter squashes. Certain types of fruitsoften referred to as pumpkin are also found in C. moshata, C. maxima,and C. mixta. Regardless of species, “pumpkin” usually refers to thefruit of the genus Cucurbita. Halloween type pumpkins (exclusivelyCucurbita pepo) can be used as a table vegetable or in pies, but areused most commonly used exclusively for autumn decoration. White, red,and gray varieties of pumpkin fruit are also available. Additionally,pumpkin seeds, either hulled or hull-less, can be roasted and consumed.Pumpkin seeds are a good source of iron and minerals.

Botanically, the fleshy, edible portion of the pumpkin is a fruit, butis conventionally considered a vegetable. The pumpkin fruit may be verylarge or small, but are typically grown to the mature stage. Pumpkinsthat are grown to maturity for use as Jack-o-lanterns or as pieingredients often have a stringy coarse flesh that varies in color fromorange to yellow to white. The stems of the genus Cucurbita usually helpidentify the species. For example, C. pepo and C. mixta have hard,five-angled stems, but C. moschata has a long, slender, columnarfive-angled stem, and C. maxima has a soft, round stem. The leaves ofCucurbita are simple with 3-5 lobes.

Cucurbita plants generally have bright yellow monoecious flowers. Likestem morphology, peduncle morphology may help identify the differentspecies. C. pepo typically have a 5-8 ridged peduncle with deep grooves;C. maxima typically have a cylindrical peduncle without any grooves; C.moschata and C. mixta typically have five-ridged peduncles that flare atthe fruit attachment point, but the C. mixta peduncle is typicallyrounded and only slightly flared. All Cucurbita spp. typically haveseparate male and female flowers, requiring a pollinizer to enable fruitand seed development. Cross-pollination is possible between some pairsof the different Cucurbita species, though may be very difficult.Cucurbita spp. have spiny, sticky pollen that requires activepollinators. In the past, most pumpkins were pollinated by squash bees,but now most commercially grown pumpkins are pollinated by honeybees orhand pollinated.

C. pepo is a diploid species, with 2n=24 chromosomes. Most commerciallygrown pumpkins are F1 hybrids. Representative varieties include “BabyBoo” (white) and “Munchkin” which are both miniature C. pepo;“Prizewinner,” and “Big Moon,” which are both jumbo C. maxima; and“Magic Lantern,” “Jackpot,” “Autumn Gold,” and “Frosty.” “Small Sugar”and “Winter Luxury” are both representative cooking varieties ofpumpkin.

Pumpkin is cultivated worldwide, though the Halloween type pumpkin isgrown widely only in the U.S. and parts of Canada. In the U.S., theprincipal pumpkin growing states include Illinois, Ohio, California,Michigan, Pennsylvania, New York, and Texas, though they are produced inevery state. Most pumpkin is direct seeded in tilled fields, buttransplants are also used. No-till production is possible, but may becomplicated by weed and disease problems. Fertilizer application to thebeds is often required, and preferably is complete before planting.Pumpkin is a warm season crop that typically matures in 70-115 daysdepending on variety and environment; giant pumpkins and some tropicallyadapted pumpkins may require 150 days or more to mature.

While breeding efforts to date have provided a number of useful pumpkinlines with beneficial traits, there remains a great need in the art fornew lines with further improved traits. Such plants would benefitfarmers and consumers alike by improving crop yields and/or quality.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a pumpkin plant of theline designated HWN 130-1039T. Also provided are pumpkin plants havingall the physiological and morphological characteristics of the pumpkinline designated HWN 130-1039T. Parts of the hot pumpkin plant of thepresent invention are also provided, for example, including pollen, anovule, scion, a rootstock, a fruit, and a cell of the plant.

The invention also concerns the seed of pumpkin line HWN 130-1039T. Thepumpkin seed of the invention may be provided as an essentiallyhomogeneous population of pumpkin seed of the line designated HWN130-1039T. Essentially homogeneous populations of seed are generallyfree from substantial numbers of other seed. Therefore, seed of line HWN130-1039T may be defined as forming at least about 97% of the totalseed, including at least about 98%, 99% or more of the seed. Thepopulation of pumpkin seed may be particularly defined as beingessentially free from hybrid seed. The seed population may be separatelygrown to provide an essentially homogeneous population of pumpkin plantsdesignated HWN 130-1039T.

In another aspect of the invention, a plant of pumpkin line HWN130-1039T comprising an added heritable trait is provided. The heritabletrait may comprise a genetic locus that is, for example, a dominant orrecessive allele. In one embodiment of the invention, a plant of pumpkinline HWN 130-1039T is defined as comprising a single locus conversion.In specific embodiments of the invention, an added genetic locus confersone or more traits such as, for example, herbicide tolerance, insectresistance, disease resistance, and modified carbohydrate metabolism. Infurther embodiments, the trait may be conferred by a naturally occurringgene introduced into the genome of the line by backcrossing, a naturalor induced mutation, or a transgene introduced through genetictransformation techniques into the plant or a progenitor of any previousgeneration thereof. When introduced through transformation, a geneticlocus may comprise one or more genes integrated at a single chromosomallocation.

In another aspect of the invention, a tissue culture of regenerablecells of a pumpkin plant of line HWN 130-1039T is provided. The tissueculture will preferably be capable of regenerating pumpkin plantscapable of expressing all of the physiological and morphologicalcharacteristics of the line, and of regenerating plants havingsubstantially the same genotype as other plants of the line. Examples ofsome of the physiological and morphological characteristics of the lineHWN 130-1039T include those traits set forth in the tables herein. Theregenerable cells in such tissue cultures may be derived, for example,from embryos, meristems, cotyledons, pollen, leaves, anthers, roots,root tips, pistil, flower, seed and stalks. Still further, the presentinvention provides pumpkin plants regenerated from a tissue culture ofthe invention, the plants having all the physiological and morphologicalcharacteristics of line HWN 130-1039T.

In yet another aspect of the invention, processes are provided forproducing pumpkin seeds, plants and fruit, which processes generallycomprise crossing a first parent pumpkin plant with a second parentpumpkin plant, wherein at least one of the first or second parentpumpkin plants is a plant of the line designated HWN 130-1039T. Theseprocesses may be further exemplified as processes for preparing hybridpumpkin seed or plants, wherein a first pumpkin plant is crossed with asecond pumpkin plant of a different, distinct line to provide a hybridthat has, as one of its parents, the pumpkin plant line HWN 130-1039T.In these processes, crossing will result in the production of seed. Theseed production occurs regardless of whether the seed is collected ornot.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent pumpkin plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent pumpkin plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the male portions of flowers, (i.e., treating ormanipulating the flowers to produce an emasculated parent pumpkinplant). Self-incompatibility systems may also be used in some hybridcrops for the same purpose. Self-incompatible plants still shed viablepollen and can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same line.

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent pumpkin plants. Yet another step comprisesharvesting the seeds from at least one of the parent pumpkin plants. Theharvested seed can be grown to produce a pumpkin plant or hybrid pumpkinplant.

The present invention also provides the pumpkin seeds and plantsproduced by a process that comprises crossing a first parent pumpkinplant with a second parent pumpkin plant, wherein at least one of thefirst or second parent pumpkin plants is a plant of the line designatedHWN 130-1039T. In one embodiment of the invention, pumpkin seed andplants produced by the process are first generation (F₁) hybrid pumpkinseed and plants produced by crossing a plant in accordance with theinvention with another, distinct plant. The present invention furthercontemplates plant parts of such an F₁ hybrid pumpkin plant, and methodsof use thereof. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid pumpkin plant and seed thereof.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from line HWN 130-1039T, the method comprisingthe steps of: (a) preparing a progeny plant derived from line HWN130-1039T, wherein said preparing comprises crossing a plant of the lineHWN 130-1039T with a second plant; and (b) crossing the progeny plantwith itself or a second plant to produce a seed of a progeny plant of asubsequent generation. In further embodiments, the method mayadditionally comprise: (c) growing a progeny plant of a subsequentgeneration from said seed of a progeny plant of a subsequent generationand crossing the progeny plant of a subsequent generation with itself ora second plant; and repeating the steps for an additional 3-10generations to produce a plant derived from line HWN 130-1039T. Theplant derived from line HWN 130-1039T may be an inbred line, and theaforementioned repeated crossing steps may be defined as comprisingsufficient inbreeding to produce the inbred line. In the method, it maybe desirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, a plant derived from line HWN130-1039T is obtained which possesses some of the desirable traits ofthe line as well as potentially other selected traits.

In certain embodiments, the present invention provides a method ofproducing pumpkins comprising: (a) obtaining a plant of pumpkin line HWN130-1039T, wherein the plant has been cultivated to maturity, and (b)collecting pumpkins from the plant.

In still yet another aspect of the invention, the genetic complement ofthe pumpkin plant line designated HWN 130-1039T is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a pumpkin plant, or a cell or tissue of that plant.A genetic complement thus represents the genetic makeup of a cell,tissue or plant, and a hybrid genetic complement represents the geneticmake up of a hybrid cell, tissue or plant. The invention thus providespumpkin plant cells that have a genetic complement in accordance withthe pumpkin plant cells disclosed herein, and plants, seeds and plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that line HWN 130-1039T could be identified by any ofthe many well known techniques such as, for example, Simple SequenceLength Polymorphisms (SSLPs) (Williams et al., 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by pumpkin plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a pumpkin plant of the invention with a haploid geneticcomplement of a second pumpkin plant, preferably, another, distinctpumpkin plant. In another aspect, the present invention provides apumpkin plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the invention provides a plant of an inbredpumpkin line that exhibits a combination of traits comprising resistanceto Zucchini Yellow Mosaic Virus (ZYMV), Watermelon Mosaic Virus (WMV),and/or tolerance to Papaya Ringspot Virus (PRSV). In certainembodiments, the combination of traits may be defined as controlled bygenetic means for the expression of the combination of traits found inpumpkin line HWN 130-1039T.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of pumpkin line HWN 130-1039Tcomprising detecting in the genome of the plant at least a firstpolymorphism. The method may, in certain embodiments, comprise detectinga plurality of polymorphisms in the genome of the plant. The method mayfurther comprise storing the results of the step of detecting theplurality of polymorphisms on a computer readable medium. The inventionfurther provides a computer readable medium produced by such a method.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of pumpkin line HWN 130-1039T. This line showsuniformity and stability within the limits of environmental influencefor the traits described hereinafter. Pumpkin line HWN 130-1039Tprovides sufficient seed yield. By crossing with a distinct secondplant, uniform F1 hybrid progeny can be obtained.

Line HWN 130-1039T exhibits a number of improved traits includingresistance to ZYMV, WMV, CMV, PRSV, and Powdery Mildew caused bySphaerotheca fuliginella (Schlechtend.:Fr.) Pollacci. The development ofthe line can be summarized as follows.

A. ORIGIN AND BREEDING HISTORY OF PUMPKIN LINE HWN 130-1039T

Line HWN 130-1039T was developed by a selection strategy involving threecomponents: (1) “Magic Lantern,” a hybrid sold by Harris Moran SeedCompany having some resistance to powdery mildew caused by Sphaerothecafuliginella (Schlechtend.:Fr.) Pollacci; (2) “T42249HA,” an F4generation Seminis proprietary breeding line of Halloween type pumpkincarrying the ZW-20 transgene and also showing field tolerance to PRSV;and (3) “Merlin,” a hybrid sold by Harris Moran Seed company having someresistance to powdery mildew caused by Sphaerotheca fuliginella(Schlechtend.:Fr.) Pollacci.

T42249HA was greenhouse grown in California in the spring of 2001, whereseedlings were inoculated with ZYMV and PRSV. Survivors weretransplanted to the open field, and plants were selected from for havingthe highest level of resistance to expression of virus symptoms, and thefruit type best approximating a standard HWN type pumpkin. Pollen wascollected from a remote field plot of Merlin, and utilized forpollination in the open field of survivors from the virus screen. Seedof this F1 population was greenhouse grown during the fall of 2001 inCalifornia, where multiple plants from this population were used asmales, and crossed to Magic Lantern.

Because both the female parent of this population and the malegrandparent were hybrids, both the male parent and the resultant F1generation were segregating for a wide range of traits. To maximize theprobability of creating a breeding line with acceptable horticulturalcharacteristics, but retaining the desired level of disease resistance,the F1 generation was grown without selection. Five F1 plants wereself-pollinated in the greenhouse during the early spring of 2002 inCalifornia. The cross of (Magic Lantern X (T42249HA X Merlin)) was oneof 8 similar crosses involving T42249HA and two separate competitorvarieties, resulting in 34 separate F2 populations with a similarstructure.

These 34 F2 populations were subjected to multiple virus screens in thefall of 2002, where one population showed a particularly high level ofresistance to PRSV, and in mid-October of 2002, 6 survivors weretransferred to a greenhouse in California. One of these plants wassuccessfully self pollinated resulting in an F3 generation, which wasgreenhouse grown in the spring of 2003, and inoculated with ZYMV andPRSV. Survivors of this screen were transplanted to the open field inCalifornia, where the plants showed a uniform and high level ofresistance to PRSV. Two individuals of the F3 generation were selfpollinated. The resulting F4 generation was greenhouse grown in Augustof 2003, where seedlings were inoculated with ZYMV and PRSV, to verifythe level and uniformity of resistance. Additionally, a test cross wasperformed concurrently with self pollination.

Beginning in the F5 generation, evaluation of these breeding lines wasbased on both performance of the breeding lines and performance ofhybrids created using these breeding lines. For the line eventuallydesignated HWN 130-1039T, the F5 generation was self pollinated duringthe 2004 summer in an open field in California, concurrently with theproduction of 2 test hybrids. The F6 generation was greenhouse grown inthe spring of 2005, where it was self pollinated. These additionalgenerations further establish uniformity of the parental line, and thefounding stock of HWN 130-1039T was based on a bulk of seed from 6plants in the F7 generation, all from the same F6 generation source. Thepedigree and lineage of the F7 generation bulk is(MagicLantern/(T42249HAXMerlin))-1-1-2-3-1-M.

HWN 130-1039T has a complement of resistance traits and horticulturalcharacteristics that make it ideal for many purposes, including forproduction of Halloween type pumpkin hybrids adapted to the UnitedStates, especially the southeastern region where PRSV prevalence limitsproduction. HWN 130-1039T may be used in the development of hybrids forother markets.

B. PHYSIOLOGICAL AND MORPHOLOGICAL CHARACTERISTICS OF PUMPKIN LINE HWN130-1039T

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of pumpkin line HWN 130-1039T. A description of thephysiological and morphological characteristics of pumpkin line HWN130-1039T is presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics of Line HWN130-1039T CHARACTERISTIC HWN 130-1039T 1. General Descriptors FruitShape/Variety Group Pumpkin Expected Primary Usage Ornamental Parts ofPlant Providing Expected Mature Fruit Primary Usage Cotyledons MeasuredBetween Full Length to Width ratio—1.79 Expansion of 1^(st) and 2^(nd)True Leaves Apex—Not Notched Veining—Obvious 2. Main Stem Main ColorDark Green (Nearly Entire Length—Multipik, Jack O'Lantern, Howden) WhiteMarks at Nodes Present Yellow Marks (Associated with Absent PrecociousYellow Gene Complex) Growth Habit (20 True Leaves) Vein Moderate (SmallSugar, Spookie, Magic Lantern, Table Queen) Tendrils (20 True Leaves)Present and Elongated Main Stem Internode Dimensions Observed after20^(th) Internode Developed Length Increases from 5^(th) to 15^(th)Internode Width Decreases from 5^(th) to 15^(th) Internode 3. PetiolesDerived from Main Stem Observed after 20^(th) Node Developed Length toWidth Medial Ratio of 29.86 10th Length to Width Medial Ratio of 28.5815^(th) Petiole Spininess (Prickles) Observed after Noticeably Spiny(Early Prolific Straightneck)) 20^(th) Internode Angle of 6^(th) through15^(th) on Main Horizontal (Caserta, less than 10°) Stem 4. LaminaeLobing of 10^(th) and 15^(th) on Main Shallowly Lobed Stem Dimensions ofLeaf after 20^(th) 0.72 Length to Maximal Width Ratio of 10^(th) TrueInternode Leaf Dimensions of Leaf after 20^(th) 0.81 Length to MaximalWidth ratio of 15^(th) True Internode Leaf Silver Blotching or MottlingSilver Blotching over a Small Amount of the Surface 5. Flowers Numberper Node One (almost always) (Multipik, Cocozelle) Staminate on day ofAnthesis on Main Stem (Between Nodes 11/20) Length from Base of Calyx to102.6 mm Tip of Corolla Exterior Width at Top of Calyx 15.1 mm CupPedicel Length 392.2 mm Length of Anther Column 14.2 mm Dominant Colorof Corolla of Orange-yellow (Day of Anthesis) Staminate Flower Ring atBase of Interior of Staminate Absent Corolla Ring at Base of Interior ofPistillate Yellow Corolla Pistillate Flower on Day of Anthesis Lengthfrom Base of Calyx to 87.4 mm Tip of Corolla Pedicel Length 73.9 mmOvary Color (Day Prior to Anthesis) Green (Black Beauty, Multipik,Cocozelle, Clarita) 6. Immature Fruit Size (3-5 Days Past Anthesis)Length (through Axis) to Medial  1.56 Width Ratio Length (through Axis)to  1.34 Maximal Width Ratio Color (3-5 Days Past Anthesis) Light Green(Arlika, Clarita, Small Sugar, Ronde de Nice) Fruit Flecks Medium(Multipik, Nano Verde di Milano) Fruit Warting Absent (Cocozelle,Multipik, Ronde de Nice, Gentry) 6. Mature Fruit Surface TopographGrooving—Distinct (Howden) Warting Absent (Cocozelle, Multipik, Ronde deNice) Rind Not Lignified (when Cutting Mature Fruit, Little Cracks Form)Stylar End Nearly Flat (Multipik, True French) Peduncle End Nearly FlatSurface Neither Netted or Cracked Exterior Color Yellow-orange 7. SeedHull (Mature) Present with Normal Appearance *These are typical values.Values may vary due to environment. Other values that are substantiallyequivalent are within the scope of the invention.

Line HWN 130-1039T has been self-pollinated and planted for a number ofgenerations to produce the homozygosity and phenotypic stability to makethis line useful in commercial seed production. No variant traits havebeen observed or are expected for this line.

Pumpkin line HWN 130-1039T, being substantially homozygous, can bereproduced by planting seeds of the line, growing the resulting pumpkinplant under self-pollinating or sib-pollinating conditions andharvesting the resulting seeds using techniques familiar to one of skillin the art.

C. BREEDING PUMPKIN LINE HWN 130-1039T

One aspect of the current invention concerns methods for crossing thepumpkin line HWN 130-1039T with itself or a second plant and the seedsand plants produced by such methods. These methods can be used forpropagation of line HWN 130-1039T, or can be used to produce hybridpumpkin seeds and the plants grown therefrom. Hybrid seeds are producedby crossing line HWN 130-1039T with second pumpkin parent line.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing line HWN 130-1039T followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing with any second plant. In selectingsuch a second plant to cross for the purpose of developing novel lines,it may be desired to choose those plants which either themselves exhibitone or more selected desirable characteristics or which exhibit thedesired characteristic(s) when in hybrid combination. Once initialcrosses have been made, inbreeding and selection take place to producenew varieties. For development of a uniform line, often five or moregenerations of selfing and selection are involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with lineHWN 130-1039T and progeny thereof to achieve a homozygous line.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The line of the present invention is particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the line. In selecting a second plant to cross with HWN130-1039T for the purpose of developing novel pumpkin lines, it willtypically be preferred to choose those plants which either themselvesexhibit one or more selected desirable characteristics or which exhibitthe desired characteristic(s) when in hybrid combination. Examples ofdesirable traits of pumpkins include: high seed yield, high seedgermination, seedling vigor, early fruit maturity, high fruit yield,ease of fruit setting, disease tolerance or resistance, and adaptabilityfor soil and climate conditions. Consumer-driven traits, such as apreference for a given fruit size, shape, color, texture, and taste, areother traits that may be incorporated into new lines of pumpkin plantsdeveloped by this invention.

Particularly desirable traits that may be incorporated by this inventionis improved resistance to different viral, fungal, and bacterialpathogens. Downey mildew and Phytophthora blight are fungal diseasesaffecting various species of pumpkin. Fruit and leaf lesions and fruitrot are the commercially important aspects of these diseases. Bacterialleaf spot and bacterial wilt are other diseases affecting pumpkinplants, especially during a wet season. Viral pathogens affectingpumpkin plants include the Cucurbit Leaf Crumple Virus and the CucumberMosaic Virus.

Improved resistance to insect pests is another desirable trait that maybe incorporated into new lines of pumpkin plants developed by thisinvention. Insect pests affecting the various species of pumpkin includethe Striped cucumber beetle, the Squash bug, aphids, and mites.

D. PERFORMANCE CHARACTERISTICS

As described above, line HWN 130-1039T exhibits desirable agronomictraits, including resistance to ZYMV, WMV, and PRSV. In addition,hybrids made using HWN 130-1018T as a parent line also demonstratemultivirus resistance, along with other desirable horticultural traits.Hybrids made with the line were the subject of objective analysis ofperformance traits of hybrids made using HWN 130-1018T compared to otherSeminis Halloween type pumpkin products. The results of the analysis arepresented below in Table 2. During the year and at the location wherethis analysis was performed, natural incidence of viral diseases wasunusually low, so that resistance levels for ZYMV, WMV, and PRSV werenot observable in this trial. As the truly unique and valuable featureof hybrids derived from HWN 130-1018T is their multivirus resistance,the horticultural characteristics of these lines need only match thoseof a typical commercial product to demonstrate value to the farmer.

TABLE 2 Horticultural Scoring Of Halloween Type Pumpkins Produced UsingHWN 130- 1039T as a Parent Compared To Those Not Produced Using HWN130-1039T HWN 130- 1039T Handle Handle Handle Fruit Avg Fruit CultivarHybrid DTM PM Res color length width Uniformity Plant Habit wt PXT13024949 III No 69 8 5 5 5 3 3 15.7 PXT 13035718 III Yes 78 3 6 4 6 2 214.0 APPALACIAN No 79 8 6 7 6 5 5 22.4 HARVESTMOON No 75 8 7 6 7 3 3 8.8Longface No 76 8 4 5 7 5 5 18.1 Orange Smoothie No 75 7 5 7 7 3 1 5.7PHANTOM No 85 8 6 6 4 5 5 14.8 PS159011 No 87 7 5 7 7 4 3 1.5 PX13056567 No 78 7 4 5 5 5 5 23.5 Schooltime No 75 7 4 7 7 3 1 8.1 SpiritNo 73 8 5 6 7 3 2 12.6 Spooktacular No 75 7 7 5 6 2 5 3.4 Trickster No75 7 6 3 5 7 5 2.2

Cultivar describes the commercial or pre-commercial name of the hybridbeing evaluated. HWN 130-1039T Hybrid describes whether the cultivarbeing evaluated uses the parent in question in production of the hybrid.Remaining traits are rated on a 1 to 9 scale, with 1 being ideal and 9being completely unacceptable. DTM describes the approximate number ofdays from sowing to harvest maturity in the trial where these cultivarswere evaluated. PM res represents the level of natural infestation bythe Powdery Mildew fungus, where 1 represents complete absence ofinfestation symptoms, 3 represents a high level of resistance (minimalevidence of easily visible fungal growth), 5 represents an intermediatelevel of resistance (field tolerance, moderate fungal growth mostlylimited to older leaves), 7 represents a very low level of resistance(only the youngest leaves show temporary absence of fungal growth), and9 represents complete coverage of all aboveground tissues by fungalinfestation. Handle color describes the color intensity and uniformityon the peduncle (fruit stem) of the mature fruits, where 1 represents aneven and very dark green (near black) peduncle and 9 represents a whiteor lightly colored and mottled peduncle. Handle length describes thelength of the peduncle on the mature fruit, where 1 is averagingapproximately 1 inch or less and 9 is averaging approximately 9 inchesor more. Handle width describes the peduncle in cross section relativeto fruit size, where 1 represents a peduncle that is exceptionally wide,such as a 3 inch wide peduncle on an 18 pound fruit, and 9 represents anunacceptably narrow peduncle, such as a ½ inch wide peduncle on an 18pound fruit. Fruit uniformity describes the overall level of variationfor mature fruit size, shape, color, and weight. Plant Habit describesthe space required for each plant, where 1 represents a near bush typegrowth habit and 9 represents an extremely vigorous vining growth habit.Fruit weight is the average weight of the 10 fruits harvested nearestthe center of each plot, measured in pounds.

As shown above, the hybrid using HWN 130-1018T as a parent exhibitacceptable earliness, fruit uniformity, and growth habit when comparedto most other Seminis commercial and pre-commercial Halloween typepumpkin products, and much improved resistance for Powdery Mildew. Oneimportant aspect of the invention thus provides seed of the variety forcommercial use. Such seed can be reproduced by crossing the line underself- or sib-pollinating conditions.

E. FURTHER EMBODIMENTS OF THE INVENTION

When the term pumpkin line HWN 130-1039T is used in the context of thepresent invention, this also includes plants modified to include atleast a first desired heritable trait. Such plants may, in oneembodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique. The term single locus converted plant as used herein refersto those pumpkin plants which are developed by a plant breedingtechnique called backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single locus transferred into the varietyvia the backcrossing technique.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentalpumpkin plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental pumpkin plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a pumpkin plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny pumpkin plants of a backcross in which HWN130-1039T is the recurrent parent comprise (i) the desired trait fromthe non-recurrent parent and (ii) all of the physiological andmorphological characteristics of pumpkin line HWN 130-1039T asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

Pumpkin varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the downy mildewresistance trait. For this selection process, the progeny of the initialcross are sprayed with downy mildew spores prior to the backcrossing.The spraying eliminates any plants which do not have the desired downymildew resistance characteristic, and only those plants which have thedowny mildew resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

Selection of pumpkin plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection applicable to the breeding ofpumpkin are well known in the art. Such methods will be of particularutility in the case of recessive traits and variable phenotypes, orwhere conventional assays may be more expensive, time consuming orotherwise disadvantageous. Types of genetic markers which could be usedin accordance with the invention include, but are not necessarilylimited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).

F. PLANTS DERIVED FROM PUMPKIN LINE HWN 130-1039T BY GENETIC ENGINEERING

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the pumpkin line of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plants,including pumpkin plants, are well known to those of skill in the art(see, e.g., Schroeder et al., 1993). Techniques which may be employedfor the genetic transformation of pumpkin plants include, but are notlimited to, electroporation, microprojectile bombardment,Agrobacterium-mediated transformation and direct DNA uptake byprotoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

Agrobacterium-mediated transformation of pumpkin explant material andregeneration of whole transformed pumpkin plants (including tetraploids)from the transformed shoots has been shown to be an efficienttransformation method (U.S. Pat. No. 5,262,316).

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target pumpkin cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., 1985). Moreover, recent technological advances in vectorsfor Agrobacterium-mediated gene transfer have improved the arrangementof genes and restriction sites in the vectors to facilitate theconstruction of vectors capable of expressing various polypeptide codinggenes. The vectors described have convenient multi-linker regionsflanked by a promoter and a polyadenylation site for direct expressionof inserted polypeptide coding genes. Additionally, Agrobacteriumcontaining both armed and disarmed Ti genes can be used fortransformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., 1985; U.S. Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Omirulleh et al., 1993; Fromm et al., 1986;Uchimiya et al., 1986; Marcotte et al., 1988). Transformation of plantsand expression of foreign genetic elements is exemplified in Choi et al.(1994), and Ellul et al. (2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for pumpkin plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel et al., 1985), including monocots(see, e.g., Dekeyser et al., 1990; Terada and Shimamoto, 1990); atandemly duplicated version of the CaMV 35S promoter, the enhanced 35Spromoter (P-e35S) the nopaline synthase promoter (An et al., 1988), theoctopine synthase promoter (Fromm et al., 1989); and the figwort mosaicvirus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and anenhanced version of the FMV promoter (P-eFMV) where the promotersequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus19S promoter, a sugarcane bacilliform virus promoter, a commelina yellowmottle virus promoter, and other plant DNA virus promoters known toexpress in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can beused for expression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat (Callis et al., 1988), (2) light (e.g.,pea rbcS-3A promoter, Kuhlemeier et al., 1989; maize rbcS promoter,Schaffner and Sheen, 1991; or chlorophyll a/b-binding protein promoter,Simpson et al., 1985), (3) hormones, such as abscisic acid (Marcotte etal., 1989), (4) wounding (e.g., wunl, Siebertz et al., 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., 1987; Schernthaner et al., 1988; Bustos et al., 1989).

Exemplary nucleic acids which may be introduced to the pumpkin lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a pumpkin plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a pumpkin plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880275, hereinincorporated by reference it their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., 1991). The RNA could also be a catalytic RNA molecule (i.e., aribozyme) engineered to cleave a desired endogenous mRNA product (seefor example, Gibson and Shillito, 1997). Thus, any gene which produces aprotein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of the present invention.

G. DEFINITIONS

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor conferring malesterility or a chemical agent.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Regeneration: The development of a plant from tissue culture.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the desired morphological and physiological characteristics of apumpkin variety are recovered in addition to the characteristics of thesingle locus transferred into the variety via the backcrossing techniqueand/or by genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a pumpkin plant by transformation.

H. DEPOSIT INFORMATION

A deposit of pumpkin line HWN 130-1039T, disclosed above and recited inthe claims, has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The date ofdeposit was 30 Apr. 2007. The accession number for those deposited seedsof pumpkin line HWN 130-1039T is ATCC Accession No. PTA-8393. Uponissuance of a patent, all restrictions upon the deposit will be removed,and the deposit is intended to meet all of the requirements of 37 C.F.R.§1.801-1.809. The deposit will be maintained in the depository for aperiod of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedif necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   U.S. Pat. No. 5,262,316-   U.S. Pat. No. 5,378,619-   U.S. Pat. No. 5,463,175-   U.S. Pat. No. 5,500,365-   U.S. Pat. No. 5,563,055-   U.S. Pat. No. 5,633,435-   U.S. Pat. No. 5,689,052-   U.S. Pat. No. 5,880,275-   U.S. Pat. No. 7,087,819-   An et al., Plant Physiol., 88:547, 1988.-   Berke, J. New Seeds, 1:3-4, 1999.-   Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991.-   Bustos et al., Plant Cell, 1:839, 1989.-   Callis et al., Plant Physiol., 88:965, 1988.-   Chae et al., Capsicum Eggplant Newsltr., 22:121-124, 2003.-   Choi et al., Plant Cell Rep., 13: 344-348, 1994.-   Dekeyser et al., Plant Cell, 2:591, 1990.-   Ellul et al., Theor. Appl. Genet., 107:462-469, 2003.-   EP 534 858-   Fraley et al., Bio/Technology, 3:629-635, 1985.-   Fromm et al., Nature, 312:791-793, 1986.-   Fromm et al., Plant Cell, 1:977, 1989.-   Gibson and Shillito, Mol. Biotech., 7:125, 1997-   Klee et al., Bio-Technology, 3(7):637-642, 1985.-   Kuhlemeier et al., Plant Cell, 1:471, 1989.-   Marcotte et al., Nature, 335:454, 1988.-   Marcotte et al., Plant Cell, 1:969, 1989.-   Midwest Veg. Prod. Guide for Commercial Growers (ID:56), 2003-   Odel et al., Nature, 313:810, 1985.-   Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993.-   Pandal et al., Theor. Appl. Gene., 68(6):567-577, 1984.-   Pickersgill and Barbara, Euphytica, 96(1):129-133, 1997-   Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.-   Roshal et al., EMBO J., 6:1155, 1987.-   Schaffner and Sheen, Plant Cell, 3:997, 1991.-   Schemthaner et al., EMBO J., 7:1249, 1988.-   Siebertz et al., Plant Cell, 1:961, 1989.-   Simpson et al., EMBO J., 4:2723, 1985.-   Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990.-   Uchimiya et al., Mol. Gen. Genet., 204:204, 1986.-   Wang et al., Science, 280:1077-1082, 1998.-   Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990.-   WO 99/31248

1. A seed of pumpkin line HWN 130-1039T, a sample of seed of said linehaving been deposited under ATCC Accession Number PTA-8393.
 2. A plantgrown from the seed of claim
 1. 3. A plant part of the plant of claim 2.4. The plant part of claim 3, wherein said part is selected from thegroup consisting of a fruit, pollen, rootstock, scion, an ovule and acell.
 5. A pumpkin plant, or a part thereof, having all thephysiological and morphological characteristics of the pumpkin plant ofclaim
 2. 6. A tissue culture of regenerable cells of pumpkin line HWN130-1039T, a sample of seed of said line having been deposited underATCC Accession Number PTA-8393.
 7. The tissue culture according to claim6, comprising cells or protoplasts from a plant part selected from thegroup consisting of embryos, meristems, cotyledons, pollen, leaves,anthers, roots, root tips, pistil, flower, seed and stalks.
 8. A pumpkinplant regenerated from the tissue culture of claim 6, wherein theregenerated plant expresses all of the physiological and morphologicalcharacteristics of pumpkin line HWN 130-1039T, a sample of seed of saidline having been deposited under ATCC Accession Number PTA-8393.
 9. Amethod of producing pumpkin seed, said method comprising crossing theplant of claim 2 with itself or a second pumpkin plant.
 10. The methodof claim 9, wherein the plant of pumpkin line HWN 130-1039T is thefemale parent.
 11. An F1 hybrid seed produced by the method of claim 9.12. An F1 hybrid plant produced by growing the seed of claim
 11. 13. Amethod for producing a seed of a line HWN 130-1039T-derived pumpkinplant said method comprising the steps of: (a) crossing a pumpkin plantof line HWN 130-1039T, a sample of seed of said line having beendeposited under ATCC Accession Number PTA-8393, with a second pumpkinplant; and (b) allowing seed of a HWN 130-1039T-derived pumpkin plant toform.
 14. The method of claim 13, further comprising the steps of: (c)crossing a plant grown from said HWN 130-1039T-derived pumpkin seed withitself or a second pumpkin plant to yield additional HWN130-1039T-derived pumpkin seed; (d) growing said additional HWN130-1039T-derived pumpkin seed of step (c) to yield additional HWN130-1039T-derived pumpkin plants; and (e) repeating the crossing andgrowing steps of (c) and (d) to generate further HWN 130-1039T-derivedpumpkin plants.
 15. A method of vegetatively propagating a plant ofpumpkin line HWN 130-1039T said method comprising the steps of: (a)collecting tissue capable of being propagated from a plant of pumpkinline HWN 130-1039T, a sample of seed of said line having been depositedunder ATCC Accession Number PTA-8393; (b) cultivating said tissue toobtain proliferated shoots; and (c) rooting said proliferated shoots toobtain rooted plantlets.
 16. The method of claim 15, further comprisinggrowing plants from said rooted plantlets.
 17. A method of introducing adesired trait into pumpkin line HWN 130-1039T said method comprising:(a) crossing a plant of line HWN 130-1039T, a sample of seed of saidline having been deposited under ATCC Accession Number PTA-8393, with asecond pumpkin plant that comprises a desired trait to produce F1progeny; (b) selecting an F1 progeny that comprises the desired trait;(c) crossing the selected F1 progeny with a plant of line HWN 130-1039T,a sample of seed of said line having been deposited under ATCC AccessionNumber PTA-8393, to produce backcross progeny; (d) selecting backcrossprogeny comprising the desired trait and the physiological andmorphological characteristic of pumpkin line HWN 130-1039T; and (e)repeating steps (c) and (d) three or more times in succession to produceselected fourth or higher backcross progeny that comprise the desiredtrait.
 18. A pumpkin plant produced by the method of claim
 17. 19. Amethod of producing a plant of pumpkin line HWN 130-1039T, a sample ofseed of said line having been deposited under ATCC Accession NumberPTA-8393, comprising an added desired trait, the method comprisingintroducing a transgene conferring the desired trait into a plant ofpumpkin line HWN 130-1039T.
 20. A plant that comprises all of thephysiological and morphological characteristics of pumpkin line HWN130-1039T, a sample of seed of said line having been deposited underATCC Accession Number PTA-8393.
 21. A seed that produces the plant ofclaim
 20. 22. A method of determining the genotype of the plant of claim2, said method comprising obtaining a sample of nucleic acids from saidplant and detecting in said nucleic acids a plurality of polymorphisms.23. The method of claim 22, further comprising the step of storing theresults of the step of detecting the plurality of polymorphisms on acomputer readable medium.
 24. A method of producing pumpkins said methodcomprising: (a) obtaining the plant of claim 2, wherein the plant hasbeen cultivated to maturity; and (b) collecting pumpkins from the plant.